Abstract Understanding changes to the molecular features of intestinal epithelial progenitors (IEPs) and their resident niche that occur in response to a variety of environmental and genetic insults will greatly enhance our knowledge of gut disease pathogenesis. For instance, what mechanisms result in the alteration of the normal controls for IEP proliferation during normal, physiologic wound healing? The goal of this application is to build on our recent findings that during colonic epithelial injury, the indigenous population of microbes in the gastro-intestinal tract (`microbiota') activates macrophages through toll-like receptors (TLR) that in turn appear to interact with the overlying IEPs and stimulate proliferation. Microarray analysis of laser-capture microdissected mesenchymal cells reveals a number of potential molecular mediators of the epithelial regeneration including secreted factors that stimulate proliferation and maintain epithelial basement membrane integrity. Mice with loss of function for one of these genes, prostaglandin-endoperoxide synthase 2-/- (Ptgs2) mice exhibited a profound inhibition of epithelial proliferation and cellular organization within rectal crypts when injured (similar to Myd88-/- that are defective for most TLR signaling). Exogenous addition of dimethyl-prostaglandin E2 rescued the effects of this injury in both Ptgs2-/- and Myd88-/-, indicating that Myd88 signaling is upstream of Ptgs2/PGE2. In WT and Myd88-/- mice, Ptgs2 was expressed in scattered mesenchymal cells. Surprisingly, Ptgs2 expression was not regulated by injury. Rather, in WT mice, the combination of injury and Myd88 signaling led to the repositioning of a subset of the Ptgs2-expressing stromal cells (PSCs) from the mesenchyme surrounding the middle and upper crypts to an area surrounding the crypt base adjacent to ColEPs. These findings demonstrate that Myd88 and prostaglandin signaling pathways interact to preserve epithelial proliferation during injury using a previously undescribed mechanism requiring proper cellular mobilization within the crypt niche. The goals of this proposal are to test components of our core hypothesis that macrophage derived factors drive the migration of prostaglandin synthase 2 (Ptgs2) expressing mesenchymal stromal cells (PSCs) into the regions surrounding the crypt base. This movement places PSCs in a strategic position for interaction with macrophages and the regenerating colonic epithelium. Project Narrative The goal of this project is to determine the role of macrophages and mesenchymal stem cell in colonic injury. The findings from these studies should impact our understanding of the pathogenesis of human diseases such as inflammatory bowel disease where wound healing is abnormal. In the longer term, we propose that these studies will provide the basis to develop cell-based therapeutics (using mesenchymal stem cells) for these diseases.